U.S. patent number 4,018,530 [Application Number 05/633,010] was granted by the patent office on 1977-04-19 for fluorescence spectrometry employing excitation of bleaching intensity.
This patent grant is currently assigned to Block Engineering, Inc.. Invention is credited to Tomas Hirschfeld.
United States Patent |
4,018,530 |
Hirschfeld |
April 19, 1977 |
Fluorescence spectrometry employing excitation of bleaching
intensity
Abstract
An improved system is desired for examining fluorescent
material. The method comprises the steps of illuminating material
with radiation at a fluorescent excitation wavelength of said
material and at an intensity sufficient to cause bleaching of the
material; detecting over a time interval commencing with initial
illumination of the material, fluorescent emission produced by the
material during bleaching of the latter by the radiation; and
integrating over the interval the fluorescent emission detected
during the bleaching of the material. Means are provided for
illuminating the material to cause bleaching, for detecting
fluorescent emission produced during bleaching, for measuring the
decay time interval, and for integrating a signal from the
detecting means over a time interval.
Inventors: |
Hirschfeld; Tomas (Framingham,
MA) |
Assignee: |
Block Engineering, Inc.
(Cambridge, MA)
|
Family
ID: |
24537907 |
Appl.
No.: |
05/633,010 |
Filed: |
November 18, 1975 |
Current U.S.
Class: |
356/317; 356/326;
250/459.1 |
Current CPC
Class: |
G01N
21/6408 (20130101) |
Current International
Class: |
G01N
21/64 (20060101); G01J 003/30 () |
Field of
Search: |
;356/85,98
;250/458-461 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Microscope Phase Fluorometer for Determining the Fluorescence
Lifetimes of Fluorochromes; Venetta; Review of Sci. Inst.; June
1959, pp. 450-451..
|
Primary Examiner: McGraw; Vincent P.
Attorney, Agent or Firm: Schiller & Pandiscio
Claims
What is claimed is:
1. Method of examining fluorescent material, said method comprising
the steps of
illuminating said material with radiation at a fluorescent
excitation wavelength of said material and at an exposure
sufficient to cause bleaching of said material;
detecting over a time interval commencing with initial illumination
of said material, fluorescent emission produced by said material
during bleaching of the latter by said radiation; and
integrating over said interval the fluorescent emission detected
during the bleaching of said material.
2. Method as defined in claim 1 wherein said interval is
substantially the time required for the initial intensity of said
fluorescent emission has decayed to a value of about l/e times the
initial intensity, where e is the natural logarithmic base.
3. Method of determining the difference in quantum efficiency
between two fluorescent dyes in a mixture, said method comprising
the steps of:
illuminating a sample of said mixture with radiation at an
excitation wavelength of said first dye and at an exposure
sufficient to cause bleaching of said first dye;
detecting over a time interval commencing with initial illumination
of said first dye, fluorescent emission produced by bleaching of
said first dye by said radiation;
measuring the time interval required for the initial intensity of
fluorescent emission from the illuminated first dye to decay, to
some predetermined fraction thereof;
illuminating said sample of said mixture with radiation at an
excitation wavelength of said second dye and at an exposure
sufficient to cause bleaching of said second dye;
detecting over a time interval commencing with initial illumination
of said second dye, fluorescent emission produced by bleaching of
said second dye by said radiation;
measuring the time interval required for the initial intensity of
fluorescent emission from the illuminated second dye to decay to
said predetermined fraction thereof; and
comparing said time intervals required for the initial intensities
of fluorescent emission from said dyes to decay.
4. Method as defined in claim 3 wherein said two dyes are different
states of a fluorochrome dye and said states are respectively a
bound state wherein said fluorochrome dye is bound to a selected
substrate material and an unbound state wherein said fluorochrome
dye is free of binding to said material.
5. Method as defined in claim 1 wherein said step of integrating is
continued until the intensity of said fluorescent emission has
decayed to a predetermined level to thereby obtain an integrated
value, said method including the step of then obtaining a ratio
between the initial instantaneous intensity of said emission and
said integrated value.
6. Method of determining the amount of fluorescent material in
sample solution, said method comprising the steps of:
illuminating a known amount of said material with radiation at a
fluorescent excitation wavelength of said material and at an
exposure sufficient to cause bleaching of said known amount of
material;
detecting over a time interval commencing with initial illumination
of said known amount of material, the fluorescent emission
intensity produced by said known amount material during bleaching
of the latter by said radiation, until said fluorescent emission
has decayed to a predetermined fraction of its initial
intensity;
integrating over said interval the fluorescent emission detected
during the bleaching of said known amount of material to obtain a
first integral;
illuminating said sample solution with radiation at said excitation
wavelength and at an exposure sufficient to cause bleaching of the
material in said solution;
detecting over a second time interval commencing with initial
illumination of said solution, fluorescent emission produced by
said material in said solution during bleaching of the latter by
said radiation, until said emission from said material in said
solution has decayed to a predetermined fraction of its initial
intensity;
integrating over said second interval the fluorescent emission
detected during the bleaching of said material in said solution, to
obtain a second integral; and
comparing said first and second integrals.
7. Method as defined in claim 6 wherein said intervals are
substantially the time required for the intitial intensity of
fluorescent emission to decay a value of about l/e times the
initial intensity, where e is the natural logarithmic base.
8. Method of examining a mixture of two fluorescent materials in
solution, which materials exhibit substantially similar fluorescent
emission band wavelengths and have different quantum efficiencies,
said method comprising the steps of
illuminating said mixture with radiation at excitation wavelengths
of said materials and with an exposure sufficient to substantially
completely bleach said fluorescent material having the higher
quantum efficiency, but not enough to bleach said fluorescent
material having the lower quantum efficiency;
thereafter exposing said mixture to radiation at said excitation
wavelength to excite into fluorescent emission said fluorescent
material having the lower quantum efficiency; and
detecting the fluorescent emission from substantially only said
fluorescent material having the lower quantum efficiency.
Description
This present invention relates to photometric systems and more
particularly to such a system for examining fluorescent materials
such as dyes and submicron sized histological particles stained
with a fluorescent stain.
A technique commonly in use involves the use of fluorescence or
luminescence spectrometry in which one or more materials, e.g.,
histological specimens dyed with a fluorescent stain are
illuminated with radiation in the excitation wavelength of the
stain so that the latter fluoresces. The parameters of the
fluorescence (e.g. intensity, decay lifetime, spectral
distribution, etc.) are then used to characterize the specimen. For
example, observing single particles seriatim as in a number of
automatic flow systems, the intensity of fluorescent emission from
each particle can be proportional to the particle size, the
distribution of emission can be related to the particle shape, etc.
In histological spectrometry, such parameters are often useful in
clinical identification.
There are however, a number of problems associated with the
technique, tending to limit its applicability. First, many dyes
exhibit very low intensity fluorescent emission in proportion to
the intensity of the excitation radiation, and so do not appear to
be suitable for use in fluorescence spectrometry. Even with high
intensity fluorescing dyes, if one increases the intensity of
excitation radiation, the dye bleaches generally at a rate
proportional to excitation intensity, typically through photolytic
decomposition. The procedures of the prior art have therefore been
practically limited to dyes which exhibit high quantum efficiencies
(i.e. the ratio of photons emitted by the dye molecule per the
number of absorbed incident photons of excitation wavelength), and
to the use of relatively low intensity excitation radiation,
thereby reducing or minimizing bleaching. In many cases, to obtain
enhanced fluorescence relative to the background, dyeing is
accomplished with fluorochrome dyes, i.e. a dye which fluoresces
with substantially greater quantum efficiency when bound to a
substrate that when present as a free due molecule in solution.
However, the increase in quantum efficiency for a dye may depend
upon the nature of the substrate, and if absent the dye is not a
fluorochrome.
Additionally, a phenomenon known as concentration quenching occurs
in fluorescence spectrometry, i.e. if a high local concentration of
dye exists (as when a histological particle, such as an organic
molecule, has a plurality of dyed molecules bound thereto in very
close proximity to one another), such multiple loading tends to
reduce the quantum efficiency of the fluorescence induced in the
bound dye molecules.
It is therefore the principal object of the present invention to
provide a method of and apparatus for obtaining a maximum possible
fluorescent signal from a particle dyed with a fluorescent stain.
Another object of the present invention is to provide a system for
examining minute particles dyed with one or more fluorescent stains
in which the effect of a change in quantum efficiency of the
fluorescent dye (for example due to concentration quenching by
multiple loading) upon the fluorescent output signal or the effect
of bleaching due to high levels of excitation illumination is
minimized or becomes immaterial. Yet other objects of the present
invention are to provide a method of examining particles which are
dyed with fluorescent dyes normally not considered useful in the
prior art for fluorescence spectrometry for lack of adequate
quantum efficiency or fluorescent intensity and to provide a novel
method of fluorescence spectrometry. Still other objects of the
present invention are to provide a system of measuring the
difference in quantum efficiency between two states of a
fluorescent material, to provide a system for discriminating
between two different quantum efficiency states of a dye, and to
provide a system for measuring the concentration of a fluorescent
material independent of the quantum efficiency of the dye. Other
objects of the present invention will in part be obvious and will
in part appear hereinafter. The invention accordingly comprises the
apparatus possessing the construction, combination of elements, and
arrangement of parts and the method comprising the several steps
and relation and order thereof, all of which are exemplified in the
following detailed disclosure, and the scope of the application of
which will be indicated in the claims.
For a fuller understanding of the nature and objects of the present
invention, reference should be had to the following detailed
descrption taken in connection with the accompanying drawing
wherein:
FIG. 1 is a block diagram of an exemplary apparatus useful in
carrying out the technique of the present invention.
According to Kirchoff's equivalence relation, the fluorescence
emission rate (i.e. probability of emission per dye molecule per
unit time) or its reciprocal, the natural fluorescence lifetime
(.tau..sub.F) (i.e. the time required for the fluorescence to decay
from its maximum I following cessation of excitation to a value of
I/e, where e is the Naperian base) is invariant when the dye
molecule is exposed to external perturbations induced for example
by increases in the local concentration of the number of
fluorescent molecules, unless such perturbations are strong enough
to produce, in an extreme case, substantial changes in the
absorption spectrum. As noted above, the effect of increases in
concentration, i.e. concentration quenching, thus reduces the
quantum efficiency of fluorescence without affecting the
fluorescence emission rate of the mean excited molecule. One may
then postulate that it is thus the lifetime (.tau..sub.L) of the
mean molecule in its excited state that is being reduced. In other
words, the quantum efficiency drops because non-radiative processes
carry away a larger fraction of the energy, and this decay
mechanism will then lower the lifetime of the excited state of the
molecule. The natural fluorescence lifetime (.tau..sub.F) being
invariant at least to the first order, the fluorescent quantum
efficiency (Q.sub.F) is then proportional to the lifetime of the
excited state. Because quantum efficiency can be defined in this
case as the ratio of the excited state lifetime to the natural
fluorescence lifetime (.tau..sub.L /.tau..sub.F), Q.sub.F is then
seen to be proportional to the reciprocal of the natural
fluorescence lifetime.
When a fluorescent molecule is studied under very high steady state
illumination (e.g. greater than 100 watts/cm.sup.2 for fluorescein)
such as will typically be required for extreme sensitivity work,
the fluorescent molecule will be repeatedly excited at very short
intervals and will spend an appreciable fraction of the time in the
excited state. Under these conditions, the susceptibility of such
an excited state to decomposition by photolysis or by other
chemical reactions becomes very important. In other words, intense
illumination tends to produce a rapidly fading fluorescent
emission, or bleaching, as the molecules decompose. The total
energy emitted by the excited molecules will then be a function of
the initial emitted fluorescent power (determined by the number of
fluorescent molecules present, the illumination intensity and the
quantum efficiency of the fluorescent molecules) and of the
decomposition lifetime of the molecule. Integration of this
function to the point of complete bleaching shows the total emitted
energy to be proportional to the product of the quantum efficiency
and the decomposition lifetime. The decomposition lifetime must
necessarily be inversely proportional to the fraction of the time
that the molecule spends in the excited state, and this fraction of
time in turn is proportional, for any given illumination intensity
to the lifetime of the molecular excited state.
Thus, if we consider the proportionality of the lifetime of the
mean molecule in its excited state (.tau..sub.L) with the bleaching
lifetime (.tau..sub.B) (i.e. the amount of time required to effect
substantially complete bleaching of a plurality of the dye
molecules under a given illumination intensity), we note that the
product of the quantum efficiency times the bleaching lifetime is a
constant. Bleaching can be considered complete when output
radiation or fluorescent emission is substantially non-detectable
or below the noise of the detection system.
Inasmuch as the total amount or number of photons which are emitted
by an excited population of a given number of particular dye
molecules is a constant, if one measures the integral of the entire
output fluorescence during the bleaching lifetime of the dye, one
obtains thereby the maximum signal that one can possibly get from
that population. Z
The present invention therefore generally is a system of examining
fluorescent materials such as histological particles, or the like,
even of submicron size, which materials are per se fluorescent
stain, and comprises the steps of first illuminating the material
with radiation at an excitation wavelength at an exposure (i.e.
intensity-time product) sufficient to cause bleaching. Time of
exposure of material to such radiation can run from a few
milliseconds to as much as a few hundred milliseconds for practical
purposes but need only be a substantial fraction (i.e. > 1/2) of
the bleaching lifetime. While the fluorescing material is exposed
to the excitational illumination, the instantaneous fluorescence
emission intensity from the material is detected and a measurement
is made of the time interval required for the fluorescent intensity
to decay during bleaching, from its initial intensity I.sub.o to
some predetermined fraction of the intensity, e.g. I.sub.o /e where
e is the Naperian base. The time interval thus measured is
proportional to .tau..sub.F and hence Q.sub.F. If desired, a value
proportional to the total emission energy, typically an integral of
the signal from the photoelectric detector over that time integral
can be obtained. This integral is proportional to the maximum
energy obtainable from the fluorescent particles.
The term "fluorescence" as used herein is intended to mean a
luminescence stimulated by radiation and emitted during
stimulation. The term "fluorescent stain" is intended to include
fluorochrome as well as fluorescent stains or dyes, where the
context so permits.
Referring now to FIG. 1 there will be shown a particle detecting
system embodying the principles of the present invention and
comprising light source 20 for producing a beam of coherent light.
Although spatial coherence is not necessary, typically light source
20 can be a laser (such as that manufactured by Spectra Physics)
which for example, provides a 10 mW output at the desired
absorption wavelength of the dyed particles. Disposed in the path
of the beam from light source 20 is shutter 22, preferably a
standard type of electrical relay operated shutter having means
defining an aperture and a blade or obturator, such as an iris,
which can expose or open the shutter aperture for intervals, for
example of 1/50 second with rise times in the nature of around 0.5
ms.
Disposed in the path of radiation traversing shutter 22 is an
optical train 24 typically comprising a 4.times. objective lens
followed by an achromat lens typically having 22 mm .phi. and a
focal length of 44 mm. Optical train 24 is intended to direct light
from source 22 traversing shutter 22 onto specimen holder 26. The
latter is intended to support a specimen containing the particles
to be examined or can be a flow cell or the like. Specimen holder
26 for example, a Beckman 1 mm quartz sample cell, is in the focal
plane of the objective of microscope 28. The latter typically has a
4.times. objective lens preceeded with a diaphragm having a pinhole
aperture of about 100 micron diameter. The microscope is also
provided with a filter for blanking out (i.e. completely absorbing)
the specific exciting wavelengths while preferably fully
transmitting the fluorescent emissions.
Disposed at the eyepiece of microscope 28 is a photodetector such
as photomultiplier tube 30, for converting the amplitude of the
light seen by microscope 28 into proportional electrical signals
such as voltages. The output of photomultiplier tube 30 is
connectable through manual switch 32 to an output display system,
here shown as a storage type cathode ray oscilloscope 34 such as
Textronix Type 546B. Both the oscilloscope 34 and shutter 22 are
connected to a manually operable electrical trigger 36 which when
actuated provides a pulse which simultaneously initiates operation
of shutter 22 so that the latter makes, for example a 1/50 second
exposure, and enables oscilloscope 34 to store the signal from
photomultiplier tube 30. The trace presented on the face of storage
oscilloscope 34 can readily be permanently recorded, as by camera
38.
The output of photomultiplier tube 30 is also connectable through
switch 32 to an electrical integrating circuit for integrating the
output of detector 30 over a variable time period which is a
function of the initial intensity of fluorescent radiation from
sample holder 36. The electrical integrating circuit in the form
shown comprises a known sample-and-hold circuit 40 connected to the
output of switch 32 and also connected to trigger 36 so as to be
actuated by the latter so as to sample the output of detector 30
immediately following opening of shutter 22. The electrical
integrating circuit also includes known comparator 42 having one
input connected to the output of detector 30 through switch 32 and
another input connected to the output of sample-and-hold circuit
40. Comparator 42 is intended to provide an output signal which has
an amplitude dependent on the ratio of the input signal magnitude
from detector 30. The output of comparator 42 is connected as an
input to known thresholding amplifier 44. The latter typically
provides a signal output only when the signal at its input has
risen above a certain threshold value, in this case preferably when
the comparator indicates that the amplitude of the signal from
circuit 40 is e times the amplitude of the signal from detector
30.
The output of detector 30 is also connected through switch 32 to
the input of switch 46. The output of the latter is connected to
the input of integrator 48, typically an integrating operational
amplifier. The output of the latter is connected to display means
such as meter 50, a line printer or the like. Switch 46 is
connected to the output of thresholding amplifier 44 so as to be
turned "on" by a signal from the latter and is also connected to
the trigger 36 so as to be turned "off" by a pulse from the
latter.
The material, the examination of which is contemplated by the
present invention, can be any fluorescent material or a substance
or histological particle capable of having a fluorescent dye
coupled therewith, whether by direct convalent chemical bonding, by
coupling through an intermediate structure, by adsorption of the
like. Such particles would then include, but certainly not be
limited to, complex organic molecules such as enzymes, toxins,
proteins, polysaccharides, lipo-proteins, and the like; whole or
parts of micro-organisms such as bacteria, viruses, protozoa, and
the like, both live and dead; histological specimens such as cells,
cell sections, mitochondria, cellular nuclei and the like; and
inorganic materials such as metallic ions, ligands, molecular
clusters and the like.
All tagging or dyeing of the particles is accomplished with
fluorescent dye molecules, e.g. either a dye which is per se
capable of fluorescent emission when excited directly by radiation
in an absorption band, or a fluorochrome dye, i.e. a dye which
fluoresces with a substantially greater quantum efficiency when
bound to a particle than when present as a free dye molecule.
Because as noted, the quantum efficiency of the dye is not a
limiting factor, the present invention can make use of many dyes
which have such low quantum efficiencies and therefore fluoresce so
weakly, that heretofore they have not found utility in fluorescence
spectrometry. Among the fluorescent dyes which are useful in the
present invention are the following:
______________________________________ Acid Violet 4BL (C.I. No.
42575) Acridine Brilliant Orange (C.I. No. 46005) Acridine Orange
(C.I. No. 46005) Acridine Yellow (C.I. No. 56025) Acriflavine (C.I.
No. 46000) Auramine O (C.I. No. 41000) Aurophosphine G (C.I. No.
46035) Benzo Flavine (C.I. No. 46035) Berberine Sulfate (C.I. No.
75160) Brilliant Phosphine (C.I. No. 46035) Brilliant Sulfo Flavine
(C.I. No. 56205) Chrysoidine (C.I. No. 11270) Coerulein S (C.I. No.
45510) Coriphosphine O (C.I. No. 46020) Coriphosphine Fuchsin (C.I.
No. 42755) Euchrysine 2G (C.I. No. 46040) Euchrysine 3 RX (C.I. No.
46005) Flavo Phosphine R. (C.I. No. 46035) Fluorescein (C.I. No.
45350) Geranine G (C.I. No. 14930) Methylene Blue (C.I. No. 52015)
Morin (C.I. No. 75660) Neutral Red (C.I. No. 50040) Orange G (C.I.
No. 16230) Phosphine 3R (C.I. No. 46045) Primuline (C.I. No. 49000)
Pyronin GS (Pyronin extra) (C.I. No. 45005) Rhoduline Orange (C.I.
No. 46005) Rhoduline Violet (C.I. No. 29100) Rosole Red B (C.I. No.
43800) Safranin (C.I. No. 50210) Scarlet R (C.I. No. 26105) Sulpho
Rhodamine B (C.I. No. 45100) Tartrazine O (C.I. No. 19140) Thiazine
Red R (C.I. No. 14780) Thiazol Yellow (C.I. No. 19540) Thioflavine
S. (C.I. No. 49010) Thionin (C.I. No. 52000)
______________________________________
In many instances, the dyes will bond directly to a particle of
specified nature, as well known in the art. In other instances,
where the dyes will not bond or couple directly with a particular
particle, or where it is desired to load a particular particle with
more dye molecules than there are bonding sites, or where the
multiple loading of a particle by dye molecules will cause
quenching, it may be desirable to load an intermediate or carrier
molecule, such as a long chain polymer, and then bond the
dye-loaded polymer to the particle. Examples of molecules to which
there have been covalently attached a large number of fluorescent
dye molecules through a polymeric backbone are described in
copending application Ser. No. 535,095, filed Dec. 20, 1974.
Particularly, the latter patent application describes an antibody
having coupled thereto a polymeric chain having in turn a
multiplicity of fluorescent molecules coupled to the chain, without
substantially impairing the specificity of the antibody. Typically,
intermediates or carriers are polymeric molecules having reactive
sites dispersed along the length of the chain, with a chemically
different reactive site at the end of the chain. Such carrier or
intermediate molecules typically can comprise polyethyleneimines,
for example of molecular weight in the range of 1200-60,000;
polypeptides such as polylysines; polyamides, such as nylon 6;
polymeric carboxylic acids; and the like. A technique for dyeing
such carriers and for coupling them to particles is described in
said patent application Ser. 535,095. As earlier noted, the
invention permits the use of fluorescence spectrometry of weakly
fluorescent dyes such as but not limited to erythrosin,
fluorochrome dyes not bound to a sensitizing substrate, quenched
dyes, antifluorochrome dyes and the like.
In operation of the apparatus of FIG. 1, samples of suitable dyed
particles are exposed to radiation of bleaching intensities and the
intensity of the resultant fluorescent signal detected and
monitored over a variable time period established from initial
emission to a time when the intensity has decayed to a
predetermined fraction of its original value. For example, the
signal is displayed and observed on oscilloscope 34 along a
horizontal time axis appropriately time calibrated. The initial
intensity is observed and then the intensity after a limited period
of time is observed. From the intensity increment of decay and the
time required for that decay increment to occur, the bleaching
lifetime .tau..sub.B (arbitrarily established as the time required
for the initial intensity I to decay to I/e) can be readily
deduced, although the bleaching lifetime of course can also be
defined, If one wishes, as any multiple or submultiple of I/e
recognized by those skilled in the art.
To integrate total emission during .tau..sub.B, assuming that the
oscilloscope trace is long enough, the point on the time axis at
which the initial intensity I.sub.o has fallen to I/e (i.e.
I.sub.t) is determined and the area under the curve between Io and
I.sub.t, is then measured. It will also be recognized that the
integral of the decay curve is the mirror image of the latter so
that the integral can either be directly measured or can readily be
computed from the decay curve.
If the oscilloscope trace is too short, then one may use the
well-known decay equation (valid for single decay mode only):
(where I.sub.o = initial intensity at a starting time t.sub.o
By measuring I.sub.o, I.sub.t and t one can solve for K and hence
the value of .tau..sub..beta..
Alternatively, automatic integration is achieved as follows. Switch
32 is closed to connect sample-and-hold circuit 40 to the output of
detector 30. Immediately after trigger 36 is activated to open
shutter 22, sample-and-hold circuit 40 is enabled to read the
output voltage from detector 30. If desirable, a time increment can
be introduced between opening of shutter 22 and enablement of
sample-and-hold circuit 40 by introduction of an appropriately
timed delay line into the input to circuit 40. Circuit 40 thus
samples the initial and maximum amplitude I.sub.m of the voltage
output from detector 30 and holds that voltage at one input of
comparator 42 at a substantially constant level. The voltage at the
other input to comparator 40 is the time decaying voltage I.sub.t
from detector 30. Hence, the output of comparator is proportional
to the ratio I.sub.m /I.sub.t and when I.sub.t has decayed so that
the ratio reaches an arbitrary value (for example here the value
e), thresholding amplifier 44 is actuated to produce an output
pulse.
The activation of trigger 36 also closes switch 46 to connect the
output of detector 30 to integrating amplifier 48, and the output
pulse from amplifier 44 opens switch 46, terminating the
integration (and also clearing circuit 40). Hence it will be seen
that the integration performed by amplifier 48 is over a time
period which is variable in accordance with the initial amplitude
of the fluorescence seen by detector 30. The integral obtained can
be displayed or otherwise further processed in meter 50.
Operation of the system of FIG. 1 to establish relative
independence of signal from quantum efficiency is described in the
following examples, in each of which the sample containing the dyed
particles is irradiated in an absorption band of the dye by light
source 20 and the fluorescence from the continuously irradiated
sample is detected at one or more peak emission wavelengths by
photomultiplier 30 which converts the input light intensity to a
corresponding voltage. The output of photomultiplier 30, as noted,
can either be stored and displayed as a continuous trace of
intensity against time on oscilloscope 32 for a bleaching lifetime
computed from the oscilloscope data and th desired integral then
determined, or can be directly integrated over a bleaching lifetime
which is automatically determined.
EXAMPLE I
Polyethyleneimine molecules are dyed with fluorescein as
follows:
The fluorescein can be functionalized by the known technique of
nitrating with HNO.sub.3 and reducing the nitrate with nascent
hydrogen produced by adding HCl and Zn, thiophosgene being then
added to form fluorescein isothiocyanate. However, fluorescein
isothiocyanate is also available commercially.
To an aqueous solution of 2 mg of polyethyleneimine (PEI) (mol. wt.
20,000) in 1 ml. of 0.1M sodium cacodylate at pH 7.0 is added 50
mg. of fluorescein isothiocyanate in 1.5 ml. of water. The mixture
is stirred continuously for about 16 hours during which time light
is excluded. Excess dye is them removed by passage through a
Sephadex G-25 (silica gel) column (0.9 .times. 30 cm) and
subsequent elution of the column with 0.1 M, pH 7.0 aqueous sodium
cacodylate.
The resulting polymer/dye complex can be analyzed by the
Folin-Ciocaulteau protein assay. That assay gives a linear curve
with polyethylenimine and thus is suitable for estimation of the
amount of polymer present. The extinction coefficient of
fluorescein isothiocyanate at 495 nm. is 73 .times. 10.sup.3 and
drops to 75% of this value on binding. By measuring both polymer
and dye present in a given sample of the complex, the degree of dye
binding is estimated. This degree of binding depends upon the dye
concentration in the initial reaction mixture. The complex prepared
by the process of this Example contains approximately 80 dye
molecules per molecule of PEI.
Assuming the quantum efficiency of a pure fluoresceine solution at
a concentration of 5 ppm to be 100%, absorption measurement at 524
.mu. established that the quantum efficiency of the dyed polymer of
this example was 1.79%. The product of quantum efficiency times
bleaching time is therefore 145.
The dyed polymer solution was diluted with pure water to a
20.times. dilution and a sample placed in a 1 mm Beckman quartz
sample cell 26. The sample was illuminated by laser 20 with an
excitation wavelength of 4880 A and at an illumination intensity of
1.24 .times. 10.sup.4 w/cm.sup.2, fluorescence at th peak emission
wavelength of 524 .mu. was detected by photomultiplier tube 30 and
appeared on scope 34 as a 60 mv value. After 33 msec, the
fluorescent intensity at 524 .mu. appeared on the scope to be 40
mv. The bleaching lifetime was then computed as 81.4 msec. allowing
about 0.25 msec as a correction for operation of the shutter
22.
EXAMPLE II
The procedure of Example I is followed however altering the molar
ratio of dye to polyethylenimine (mol. wt. 20,000) so as to provide
a polymer/dye complex containing approximately 100 molecules of dye
per molecule of polyethylenimine.
Upon bleaching, the resulting data yielded a corrected bleaching
lifetime of 117.4 msec. The quantum efficiency of the dyed PEI in
this example was measured by absorption as 13.2%, a value
consistent with the increased dye loading compared to Example I.
The product of quantum efficiency times bleaching time is 155,
indicating substantial equivalence (deviation less than 7%) between
the bleaching lifetime times quantum efficiency product and thus a
first order independence from quantum efficiency.
EXAMPLE III
The polymer/dye complex of Example I was coupled to a commercially
obtained sample of Echo 12 antibody according to the procedure
described in the above-mentioned U.S. Pat. application Ser. No.
535,095 in which the PEI is first treated with glutaraldehyde (25%
aqueous) buffered to pH 7.0 prior to dyeing, the polymer/dye
complex then being directly reacted with the antibody. The
polymer/dye antibody complex in which the antibodies have coupled
thereto dye-bearing polymer molecules, was illuminated according to
Example I and the resulting data provide a bleaching lifetime of
49.5 msec and a quantum efficiency of 2.03%, the product of these
two values being 101.
EXAMPLE IV
The polymer/dye complex of Example II was coupled to a sample of
the same Echo 12 antibody according to Example III and the
resulting complex illuminated as in Example III to yield data
providing a bleaching lifetime of 88.9 msec and a Q.sub.F of 1.21%.
The lifetime .times. Q.sub.F product is 108 again exhibiting the
first order independence of the technique of the invention with
regard to Q.sub.F.
EXAMPLE V
Polyethyleneimine of mol. wt. 1200 (5% by weight in water) was dyed
with fluorescein according to Example I and samples of the dyed
polymer were diluted to provide several different concentrations.
Each was illuminated and the fluorescent output integrated
according to Example I with the following results:
______________________________________ SAMPLE BLEACH. Q.sub.F
BLEACH CONC. Q.sub.F TIME TIME
______________________________________ 100 93.7% 3.0 msec 281 333
ppm 86.0% 2.9 msec 249 1000 ppm 64.9% 4.0 msec 260 3330 ppm 30.2%
13.7 msec 414 ______________________________________
The departures from constancy of the last column, which show a
surprising signal increase at low quantum efficiencies, arise from
the departures from exponentiality towards the latter part of the
decay curve. A reduction in the decay rate in this very small
region is to be expected due to diffusion from the surroundings.
That this is the cause for the above mentioned unexpected
improvements is borne out by the lower intensity of the change for
the samples of Examples I and II on the one hand and Examples III
and IV on the other, with their much lower diffusivity. This is
consistent with the mean Brownian displacement in 10 msec, which is
about 0.7.mu. for the fluorescein and about 0.3.mu. for the
polymer. Equivalence between the products of decay time times
quantum efficiency of the samples of Example V with those of the
other Examples is not to be expected, as they correspond to
different chemical states of the dye molecule.
The first order independence from quantum efficiency of the last
column, to which the received signal per molecule will be
proportional, is clearly underscored. The total received signal
will be proportional to the produce of this number and the
loading.
As noted earlier, the principles of the present invention provide a
method for determining the difference in quantum efficiency between
two states of a fluorescent material. For example, it is often
desirable to determine the difference in the changing quantum
efficiency between the bound and unbound state of a cluorochrome
dye in order to determine how effective the dye really is as a
fluorescent source. The determination of the difference is quantum
efficiency using the principles of the present invention is quite
simple. One simply illuminates a first sample of the fluorescent
material in a first state as hereinbefore described to effect
bleaching, and detects the instantaneous fluorescent emission
produced during the bleaching process. A measurement is made of the
time interval required for the fluorescent emission from the
illuminated sample to decay for example to 1/e times the initial
intensity.
Exactly the same procedure is then followed with respect to a
sample of the material in another state, the concentration and size
of the two samples, assuming them to be solutions, being
substantially identical with respect to the fluorescent
material.
Because the bleaching lifetime .tau..sub..beta. is equal to the
product of a constant K times the Q.sub.f, it will be seen that the
ratio of the two decay lifetimes
.tau..sub..beta..sbsb.1/.tau..sub..beta..sbsb.2 is independent of
the value of K and is therefore a proportional measure of the ratio
of quantum efficiencies of the two states of the fluorescent
material.
The foregoing can be immediately appreciated from the table in
Example 5 in which it will be seen that, at least for the first
three concentrations of dyed polyethyleneimine, the bleaching
lifetimes are inversely proportional to the quantum efficiency.
Hence, for example, the ratio, 3/4, of bleaching lifetimes of the
first and third samples in Example 5 are very close to the inverse
ratio of quantum efficiencies of those two samples.
The principles of the present invention can also be employed for
example to determine an unknown concentration of known fluorescent
materials in solution. This can be determined in accordance with
the following considerations:
It will be remembered that the total fluorescent emission from each
molecule of a given species is an invariant, being proportional to
the ratio of the decomposition lifetime of the molecule to the
natrual fluorescent lifetime of the molecule. Thus, one simply
measures out a known mass of fluorescent material and dissolves it
in a small volume of solvent to provide a calibration sample. The
entire mass of fluorescent material is then illuminated with
radiation of bleaching exposure and at a fluorescent excitation
wavelength with respect to the fluorescent material. The resulting
fluorescent emission is detected and summed or integrated until its
initial intensity has decayed to some value, such as I.sub.o /e.
The integral obtained will be invariant for that amount of
fluorescent material. One now illuminates a sample volume of a
solution containing an unknown amount of the fluorescent material
with a bleaching exposure at the same excitation radiation
wavelengths, and detects and integrates the fluorescent emission
until the output level drops to I.sub.o /e. The ratio of the second
integral to the first integral will be equal to the ratio of the
unknown amount of fluorescent material in the test solution to the
known amount of fluorescent material in the calibration sample.
Obviously, measurement of the volume of the test solution will
provide the data necessary to obtain the concentration of
fluorescent material in that solution.
The principles of the present invention can also be employed, in
some cases to detect and examine a fluorescent material in a
mixture of fluorescent materials; for examples, a dye in a mixture
of dyes in solution. Where a mixture of two dyes, for example, is
of two states of the same fluorochrome dye, or is a mixed solution
of two fluorescent materials of different quantum efficiencies but
substantially identical emission band wavelengths, the concurrent
fluorescent decay of the two will be seen to be a summation of
exponential functions. The emissions curve is therefore too complex
to be described and analyzed by the simple decay equation above
delineated.
However, in accordance with the present invention, because the
quantum efficiencies of the two materials are different, one need
expose the mixture to radiation sufficient to bleach substantially
only that material with the higher quantum efficiency. Fluorescence
thereafter excited in and observed from the mixture will arise
substantially only from the fluorescent material having the weaker
quantum efficiency. This technique then permits one to separate the
two materials, and if desired, to reconstruct their individual
decay characteristics.
Since certain changes may be made in the above apparatus and method
without departing from the scope of the invention herein involved,
it is intended that all material contained in the above description
or shown in the accompanying drawing shall be interpreted in an
illustrative and not in a limiting sense.
* * * * *